Cloning of the genes that code for three major subunits of Escherichia coli polymerase III
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Cloning of the genes that code for three major subunits of Escherichia coli polymerase III
Chengxi ShiMolecular Biotechnology and
BioinformaticsUppsala University
winter,2005
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About Research Training
Period: November to December Institute: Department of Cell & Molecular Biology
Work time: 9 am to 5 pm at weekdays
Supervisor: Prof. Gerhart Wargner
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Department Information
The department is divided into 6 programs
Mikrobiologi Gerhart’s research focus on: ’riboregulator’
regulatory RNAs in bacteria
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Project
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Introduction
There exists a very stringent control of DNA replication in bacteria
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Observation: mutates all the genes that are know
to negatively control replication
DNA content only goes up 1.5 – 2 folds
A thought: the number of DNA polymerase molecules in the cell is limiting (usually 8-10 molecules per cell)
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DNA polymerase III holoenzyme has a central role in chromosomal replication
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DNA polymerase III core is composed of α, ε and θ subunits
and code by dnaE, dnaQ and holE
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So we can
mutate negtively control genes
express DNA polymerase III core
Test the DNA content
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Strategy
Use pBAD-TOPO vector to insert in order dnaE, dnaQ, holE, and with very little spacing inbtween.
PCR out the genes the primers should carry different restriction sites
PCR out the genes the primers should carry different restriction sites
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Experiment protocol and result
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Overview design primers re-streak bacteria stain
PCR plasmid miniprep
PCR product purification
enzyme cleavage enzyme cleavage
gel extraction gel extraction
ligation
transform into competent cells
colony PCR test
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Primer designing For each insert: Include translation initiation site ATG
For the first insert: Include the Shine-Dalgarno sequence
GGAA
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What is Shine-Dalgarno (SD) sequence ?
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dnaE forward: 5′- [P] – CTGACTGCAGGGAATCTGAAGATGTCTGAA
PstI dnaE reverse: 5′- TAGAATTCTACCATGGTTAGTCAAACTCCAGTTCCA
EcoRI NcoI
dnaQ forward: 5′- TACCATGGAAGTCTGACATAAATGACCGCT
NcoI
dnaQ reverse: 5′- TAGAATTCTAGGTACCTTATGCTCGCCAGAGGCAAC
EcoRI KpnI
holE forward: 5′- TAGGTACCGAGGAGATTAAGAATG
KpnI
holE reverse: 5′- TAGAATTCTTATTTAAGTTTGGGCT
EcoRI
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First several bases of mRNA form a loop
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PCR result
3500bp
dnaE PCR product
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800bp
700bp
dnaQ PCR product
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Cleavage of pBAD
PvuIIEcoRI
both
open-circular
supercoiled
linear
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Ligation (Ready-to-go T4 DNA lagase) transformation (TOP-10 chemically competent E.coli)
colony PCR
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Acknowledgement
Many thanks to Prof. Gerhart Also thank the member of the lab,
especially Klas, Cia, Shiying and Erik
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Thank you!